Metals play an important role in biological systems in regulating enzyme activity, protein structure, and cellular signaling. Metals can also have a deleterious effect when present in excess of bodily requirements or capacity to excrete and, if not usually present in cells may be highly toxic to the cell. Metals, and in particular heavy metals such as zinc (Zn.sup.2+), lead (Pb.sup.2+), barium (Ba.sup.2+) cadmium (Cd.sup.2+) and mercury (Hg.sup.2+) are also significant environmental contaminants. A large number of natural and synthetic materials are known to selectively or non-selectively bind to or chelate metals. Ion chelators are commonly used in solution for in vivo control of ionic concentrations and detoxification of excess metals, and as in vitro buffers. When bound to a fluorophore, ion chelators can often be used as optical indicators of ions.
Numerous chromophoric or fluorescent complexing agents for metals, including heavy metals, have been described (see in particular G. Guilbault, PRACTICAL FLUORESCENCE, 2nd Ed., Marcel Dekker, Inc., New York, 1990). A number of indicators whose fluorescence responds to physiological ranges of Ca.sup.2+ have been described, including our copending application BENZAZOLYLCOUMARIN-BASED ION INDICATORS, filed May 20, 1994. Many of these indicators are fluorescent derivatives of the BAPTA chelators originally described by Tsien (Tsien, BIOCHEMISTRY 19, 2396 (1980)). Examples of BAPTA-based fluorescent indicators include quin-2 (Tsien, BIOCHEMISTRY supra); fura-2, and indo-1 (U.S. Pat. No. 4,603,209 to Tsien et al., 1986); fluo-3 an rhod-2 (U.S. Pat. No. 5,049,673 to Tsien et al. 1991); FURA-RED (Molecular Probes, Inc., Eugene, OR, trademark for 1-[6-amino-2(5-oxo-2-thioxo-4-thiazolidinylidene)methyl-5-benzofuranyoxy]- 2-2-(2'-amino-5'-methyl-phenoxy)ethane-N,N,N',N'-tetraacetic acid and the tetraacetoxymethyl ester thereof, U.S. Pat. No. 4,849,362 to DeMarinis, et al. 1989). Additional BAPTA-based fluorescent indicators for Ca.sup.2+ have been described by Tsien (Intracellular Measurements of Ion Activities, ANN. REV. BIOPHYS. BIOENG., 12, 91 (1983)), and Smith et al., (J. CHEM. SOC. PERKIN TRANS. 2, 1195 (1993)). Other BAPTA-based indicators are described in Copending application REACTIVE DERIVATIVES OF BAPTA USED TO MAKE ION-SELECTIVE CHELATORS, Ser. No. 07/843,360 (filed Feb. 25, 1992) by Kuhn et al.
The complexation of Zn.sup.2+ is known to enhance the fluorescence of certain BAPTA-based indicators such as fura-2 and to shift the indicator's excitation spectrum. Although fura-2 has much higher affinity for Zn.sup.2+ than do the indicators of the present invention (dissociation constants for Zn.sup.2+ complexes of fura-2 are typically in the nM range, rather than in the .mu.M range as for the instant indicators), the higher affinity of fura-2 for Ca.sup.2+ in particular makes it difficult to measure other metals such as Zn.sup.2+ and Pb.sup.2++ in the presence of physiological concentrations of Ca.sup.2+ (10.sup.-8 to 10.sup.-5 M free Ca.sup.2+) or Mg.sup.2+ (10.sup.-4 to 10.sup.-2 M free Mg.sup.2+). Furthermore, the excitation spectra of fura-2 and its Zn.sup.2+ complex is in the ultraviolet range, where quartz optics are typically needed for detection, and where intrinsic cellular autofluorescence is generally very high. An indicator capable of detecting and quantifying Zn.sup.2+, that is also insensitive to Ca.sup.2+, is therefore needed.
The indicators described in copending application BENZAZOLYLCOUMARIN-BASED ION INDICATORS (filed May 20, 1994) are less sensitive to Ca.sup.2+ than previously described BAPTA-type indicators. However, these lower-affinity indicators are largely insensitive to heavy metal ions. Substitution at the 5' position of the BAPTA chelator by nitro is known to reduce the affinity of BAPTA-based indicators for Ca.sup.2+ (e.g. see Calcium Green.TM.-5N versus Calcium Green.TM.-1 in Haugland, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, Set 20 (1992) and Pethig, et al., CELL CALCIUM 10, 491 (1989)). However, substitution by nitro at the 5'-position of the above-described low-affinity indicators almost totally quenches the fluorescence of the free (non-complexed) indicator, even though the benzazole fluorophore is bound to the opposite ring of the BAPTA chelator.
Surprisingly, the substituted of a 5'-nitro group results in a family of indicators that respond to the complexation of a variety of heavy metal ions, including the heavy metal ions Zn.sup.2+, Pb.sup.2+, Ba.sup.2+, Cd.sup.2+, Hg.sup.2+ and La.sup.3+. This result is particularly unexpected because Pb.sup.2+, Cd.sup.2+ and Ba.sup.2+ have been reported to strongly que other BAPTA-type indicators (Haugland, MOLECULAR PROBES HANDBOOK, supra, Set 20, pg. 113 (1992)). Furthermore, the instant indicators give a distinct maximum emission intensity and a specific range of sensitivity (K.sub.d) upon binding different metal ions that could be used, in principle, to detect the concentration of more than one metal ion simultaneously. The present indicators exhibit virtually no fluorescence response to the binding of either Ca.sup.2+ or Mg.sup.2+. Consequently, substitution of a 5'-nitro on the benzazolylcoumarin-based BAPTA ion indicators yields a family of indicators that is typically not useful for measuring physiologically relevant levels of Ca.sup.2+ or Mg.sup.2+.
The compounds of the present invention possess special utilility for the detection and quantification of Zn.sup.2+ ions. The currently utilized Zn.sup.2+ ion probe N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ) has been used to detect Zn.sup.2+ in physiological studies. TSQ binds Zn.sup.2+ so strongly that it is insensitive to small changes in equilibrium levels of Zn.sup.2+. The instant compounds represent the first practical equilibrium indicators for intracellular Zn.sup.2+ that are useful in the submicromolar range.
The compounds of the present invention have significant utility as a means of detecting and quantifying certain metal ion levels in living cells, biological fluids or aqueous solutions. The relatively long wavelength excitation and emission bands of the compounds of the present invention enable their use with a variety of optical devices and require no specialized (quartz) optics, such as are required by indicators that are excited or that emit at shorter wavelengths.